Gene disruption by transposable elements is an important means for isolating useful genes and analyzing their functions. Arabidopsis has been studied as a model plant, in which T-DNA and transposons are used as insertion elements.
In the case of T-DNA, when a Ti plasmid is introduced into a plant using Agrobacterium, the T-DNA, which is carried on the Ti plasmid, is inserted into one of the plant chromosomes, thereby disrupting a gene. However, it has been reported that in gene disruption experiments using T-DNA, T-DNA is not successfully integrated in 50 to 60% of mutants, so that the T-DNA does not substantially function as a tag.
In the case of a transposon, gene disruption occurs during the process of transformation or in the subsequent process of transposition. Transposons are mutagenic genes which are ubiquitous in the genomes of animals, yeast, bacteria, and plants. Transposons are classified into two categories according to their mechanism of transposition. Class II transposons of class II undergo transposition in the form of DNA without replication. Class I transposons of class I are also called retrotransposons. Retrotransposons undergo replicative transposition through RNA as an intermediate.
Examples of class II transposons include the Ac/Ds, Spm/dSpm and Mu elements of maize (Zea mays) (Fedoroff, 1989, Cell 56, 181–191; Fedoroff et al., 1983, Cell 35, 235–242; Schiefelbein et al., 1985, Proc. Natl. Acad. Sci. USA 82, 4783–4787), and the Tam element of Antirrhinum (Antirrhinum majus) (Bonas et al., 1984, EMBO J, 3, 1015–1019). Class II transposons are widely used for gene isolation by transposon tagging. This technique utilizes a specific property of transposons. When a transposon transposes within a genome and integrates into a certain gene, the gene is physically or structurally modified, and so the phenotype controlled by the gene is changed. If a phenotypic change can be detected, the affected gene may be isolated (Bancroft et al., 1993, The Plant Cell, 5, 631–638; Colasanti et al., 1998, Cell, 93, 593–603; Gray et al., 1997, Cell, 89, 25–31; Keddie et al., 1998, The Plant Cell, 10, 877–887; Whitham et al., 1994, Cell, 78, 1101–1115). However, an untagged mutant has been reported, in which the transposon is excised during DNA transposon tagging (Bancroft et al., 1993, The Plant Cell, 5, 631–638). Transposons have a tendency to transpose in the vicinity of insertion sites within chromosomes (Bancroft and Dean, 1993, Genetics, 134, 1221–1229; Keller et al., 1993, Theor. Appl. Genet, 86, 585–588). A transposon which can transpose randomly into chromosomes is desired in order to produce disruption lines covering all possible genes. However, these transposons integrate into particular target sites. A gene disruption system using different from those described above is desired.
Class I transposons were originally identified and characterized in Drosophila and yeast. A recent study has revealed that retrotransposons are ubiquitous and dominant in plant genomes (Bennetzen, 1996, Trends Microbiol., 4, 347–353; Voytas, 1996, Science, 274, 737–738). It appears that most retrotransposons are an integratable but non-transposable unit. Retrotransposons have LTRs in the forward direction at each end, and regions encoding a gag protein constituting a virus-like particle and a reverse transcriptase pol protein between the two LTRs. RNA transcribed from a LTR promoter is reverse-transcribed by the pol protein into cDNA which is in turn inserted into a host chromosome. Transposition of a retrotransposon is performed by a protein encoded by the retrotransposon itself and there is no excision mechanism. Therefore, use of retrotransposons is an excellent gene disruption technique.
Recently, it has been reported that some retrotransposons are activated under stressful conditions, such as wounding, pathogen attack, and culture (Grandbastien, 1998, Trends in Plant Science, 3, 181–187; Wessler, 1996, Curr. Biol., 6, 959–961; Wessler et al., 1995, Curr. Opin. Genet. Devel., 5, 814–821). For example, activation of the tobacco retrotransposons Tnt1A and Tto1, and the rice retrotransposon Tos17 was found to occur under stressful conditions (Pouteau et al., 1994, Plant J., 5, 535–542; Takeda et al., 1988, Plant Mol. Biol., 36, 365–376; Hirochika et al., 1996, Proc. Natl. Acad. Sci. USA, 93, 7783–7788).
In rice, the rice retrotransposon Tos17 is activated by culture and can transpose into a gene (Hirochika et al., Proc. Natl. Acad. Sci. USA, 93, 7783–7788 (1996)). Therefore, Tos17 has been utilized as a means for mass gene disruption in rice.
In Arabidopsis thaliana, no retrotransposons having transposition activity have been isolated. It has been reported that the tobacco retrotransposon Tnt1, isolated by Grandbastien et al., is transposed in the process of introducing Tnt1 into Arabidopsis by transformation. However, it has yet to be clarified whether or not Tnt1 can be transposed into a gene (Lucas et al., 1995, EMBO J., 14, 2364–2393). It has been found that the tobacco retrotransposon Tto1 is transposed during the process of transformation into Arabidopsis (Hirochika and Kakutani, in preparation). Tto1 is also transposed in rice by culture (Hirochika et al., 1996, Plant, Cell, 8, 725–734), suggesting that Tto1 is transposable in a wide range of hosts. However, the frequency of transposition varies from line to line, and a high frequency of transposition is not necessarily reproducible.